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WO2021083363A1 - Tcr à haute affinité pour la reconnaissance de kras g12v - Google Patents

Tcr à haute affinité pour la reconnaissance de kras g12v Download PDF

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WO2021083363A1
WO2021083363A1 PCT/CN2020/125475 CN2020125475W WO2021083363A1 WO 2021083363 A1 WO2021083363 A1 WO 2021083363A1 CN 2020125475 W CN2020125475 W CN 2020125475W WO 2021083363 A1 WO2021083363 A1 WO 2021083363A1
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tcr
chain
variable domain
amino acid
exon
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彭真
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Xlifesc Ltd
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Xlifesc Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of biotechnology, and more specifically to a T cell receptor (TCR) capable of recognizing a polypeptide derived from an SSX2 protein.
  • TCR T cell receptor
  • the invention also relates to the preparation and use of the receptor.
  • TCR T cell receptor
  • TCR is the only receptor for specific antigen peptides presented on the main histocompatibility complex (MHC). This exogenous or endogenous peptide may be the only sign of abnormal cells.
  • MHC main histocompatibility complex
  • APC antigen presenting cells
  • the MHC class I and class II molecular ligands corresponding to TCR are also proteins of the immunoglobulin superfamily but have specificity for the presentation of antigens. Different individuals have different MHCs, which can present different shortcomings in a protein antigen. Peptides to the surface of the respective APC cells. Human MHC is usually called HLA gene or HLA complex.
  • KRAS gene (p21 gene), a murine sarcoma virus oncogene, is a member of the ras gene family and encodes the KRAS protein. Once the KRAS gene is mutated, it will continue to stimulate cell growth, leading to the occurrence of tumors.
  • G12V is one of the common mutation sites, and this mutant is called KRAS G12V.
  • KRAS G12V is expressed in a variety of human cancer cells, including but not limited to lung cancer, colorectal cancer, pancreatic cancer, gastric cancer, etc.
  • the short peptide VVGAVGVGK after KRAS G12V mutation is located at positions 8-16 of KRAS amino acids, and is a target for the treatment of related diseases.
  • the VVGAVGVGK-HLA A1101 complex provides a marker for TCR to target tumor cells.
  • the TCR that can be combined with VVGAVGVGK-HLA A1101 complex has high application value for tumor treatment.
  • TCR that can target the tumor cell marker can be used to deliver cytotoxic or immunostimulant to target cells, or be transformed into T cells, so that T cells expressing the TCR can destroy tumor cells, so that they can be called Adoptive immunotherapy is given to patients during the course of treatment.
  • the ideal TCR has a high affinity, so that the TCR can reside on the targeted cells for a long time.
  • the purpose of the present invention is to provide a TCR with higher affinity to the VVGAVGVGK-HLA A1101 complex.
  • Another object of the present invention is to provide a method for preparing the above-mentioned type of TCR and the use of the above-mentioned type of TCR.
  • the first aspect of the present invention provides a T cell receptor (TCR), which has the activity of binding to the VVGAVGVGK-HLA A1101 complex.
  • TCR T cell receptor
  • the alpha chain variable domain of the TCR contains at least 90% of the amino acid sequence shown in SEQ ID NO:1; preferably, at least 92%; more preferably, at least 94% (e.g., It may be an amino acid sequence with sequence homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology); and/or
  • the ⁇ -chain variable domain of the TCR contains at least 90% of the amino acid sequence shown in SEQ ID NO: 2, preferably at least 92%; more preferably, at least 94% (e.g., may be at least 91%, 92%). %, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology).
  • the TCR ⁇ chain variable domain comprises three CDR regions, CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR3 ⁇ is [3 ⁇ X1][3 ⁇ X2][3 ⁇ X3][3 ⁇ X4]GNNDMR, and [3 ⁇ X1 ] Is A or G; and/or [3 ⁇ X2] is S or V or I; and/or [3 ⁇ X3] is L or M; and/or [3 ⁇ X4] is K or Q or L or M.
  • the TCR ⁇ chain variable domain comprises three CDR regions, wherein the amino acid sequence of CDR1 ⁇ is SGHVS; and the amino acid sequence of CDR2 ⁇ is FQNEAQ.
  • the TCR ⁇ chain variable domain comprises three CDR regions, wherein the amino acid sequence of CDR3 ⁇ is: ASSSSRWEQQF or ASSSSRWEPMV.
  • the TCR ⁇ chain variable domain comprises 3 CDR regions, and the sequence of the 3 CDR regions is:
  • amino acid sequence of the variable domain of the TCR ⁇ chain is SEQ ID NO: 2.
  • the TCR ⁇ chain variable domain comprises three CDR regions, wherein the amino acid sequence of CDR1 ⁇ is DRVSQS; and the amino acid sequence of CDR2 ⁇ is IYSNGD.
  • the TCR ⁇ chain variable domain comprises three CDR regions, wherein the amino acid sequence of CDR3 ⁇ is selected from the group consisting of ASLKGNNDMR, AVMQGNNDMR, GVLKGNNDMR, GVLLGNNDMR, GVLQGNNDMR, AILKGNNDMR, AVLKGNNDMR, AVLQGNNDMR, and AVLQGNNDMR.
  • amino acid sequence of the variable domain of the TCR ⁇ chain is SEQ ID NO:1.
  • the TCR has a CDR selected from the following group:
  • the amino acid sequence of the ⁇ chain variable domain of the TCR is selected from: SEQ ID NO: 1 and 13-20; and/or the amino acid sequence of the ⁇ chain variable domain of the TCR is selected from: SEQ ID NO: 2 and 21.
  • amino acid sequence of the ⁇ chain variable domain of the TCR is selected from: SEQ ID NO: 13-20; and/or the amino acid sequence of the ⁇ chain variable domain of the TCR is selected from: SEQ ID NO: 2.
  • the TCR is selected from the following group:
  • the affinity of the TCR and the VVGAVGVGK-HLA A1101 complex is at least twice that of the wild-type TCR.
  • the dissociation equilibrium constant KD of the TCR to the VVGAVGVGK-HLA A1101 complex is ⁇ 2.15 ⁇ M;
  • the TCR is soluble.
  • the TCR is ⁇ heterodimeric TCR.
  • the TCR has an ⁇ -chain constant region sequence TRAC*01 and a ⁇ -chain constant region sequence TRBC1*01 or TRBC2*01.
  • the TCR comprises (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) And (ii) both comprise the variable domain and at least a part of the constant domain of the TCR chain.
  • the alpha chain constant region and the beta chain constant region of the TCR contain artificial interchain disulfide bonds.
  • cysteine residues forming artificial interchain disulfide bonds between the constant regions of the TCR ⁇ and ⁇ chains are substituted for one or more sets of sites selected from the following:
  • the TCR is a single-chain TCR.
  • the TCR is a single-chain TCR composed of an ⁇ -chain variable domain and a ⁇ -chain variable domain, and the ⁇ -chain variable domain and the ⁇ -chain variable domain are composed of a flexible short peptide sequence (linker )connection.
  • a conjugate is bound to the C- or N-terminus of the ⁇ chain and/or ⁇ chain of the TCR.
  • the conjugate that binds to the TCR is a detectable label, a therapeutic agent, a PK modified portion, or a combination of any of these substances.
  • the therapeutic agent that binds to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the ⁇ or ⁇ chain of the TCR.
  • the second aspect of the present invention provides a multivalent TCR complex comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present invention.
  • the third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule according to the first aspect of the present invention or the multivalent TCR complex according to the second aspect of the present invention or its complement sequence.
  • the fourth aspect of the present invention provides a vector containing the nucleic acid molecule described in the third aspect of the present invention.
  • the fifth aspect of the present invention provides a host cell containing the vector of the fourth aspect of the present invention or the nucleic acid molecule of the third aspect of the present invention integrated into the chromosome.
  • the sixth aspect of the present invention provides an isolated cell that expresses the TCR described in the first aspect of the present invention.
  • the seventh aspect of the present invention provides a pharmaceutical composition containing a pharmaceutically acceptable carrier and the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, Or the cell described in the sixth aspect of the present invention.
  • the eighth aspect of the present invention provides a method for treating diseases, comprising administering an appropriate amount of the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, or the present invention to a subject in need of treatment.
  • the ninth aspect of the present invention provides the use of the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, or the cell according to the sixth aspect of the present invention, for preparing and treating tumors
  • the tumor is a Kras G12V-positive tumor, and more preferably, the tumor is pancreatic cancer, colorectal cancer or lung cancer.
  • the T cell receptor of any one of claims 1-23, the TCR complex of claim 24, or the cell of claim 28 is used as a drug for treating tumors.
  • the tumor is a Kras G12V positive tumor, more preferably, the tumor is pancreatic cancer, colorectal cancer or lung cancer.
  • the eleventh aspect of the present invention provides a method for preparing the T cell receptor according to the first aspect of the present invention, including the steps:
  • Figure 1a and Figure 1b respectively show the amino acid sequences of wild-type TCR ⁇ and ⁇ chain variable domains that can specifically bind to the VVGAVGVGK-HLA A1101 complex.
  • Figures 2a and 2b are respectively the amino acid sequence of the alpha chain variable domain and the amino acid sequence of the beta chain variable domain of the single-chain template TCR constructed in the present invention.
  • Figures 3a and 3b are respectively the DNA sequence of the ⁇ chain variable domain and the DNA sequence of the ⁇ chain variable domain of the single-stranded template TCR constructed in the present invention.
  • Figures 4a and 4b are respectively the amino acid sequence and nucleotide sequence of the linker of the single-stranded template TCR constructed in the present invention.
  • Figure 5a and Figure 5b are the amino acid sequence and DNA sequence of the single-stranded template TCR constructed in the present invention, respectively.
  • Figure 6a and Figure 6b respectively show the amino acid sequences of the soluble reference TCR alpha and beta chains of the present invention.
  • Figures 7a-7h respectively show the amino acid sequence of the ⁇ chain variable domain of a heterodimeric TCR with high affinity for the VVGAVGVGK-HLA A1101 complex, and the mutated residues are underlined.
  • Figure 8 shows the amino acid sequence of the ⁇ chain variable domain of a heterodimeric TCR with high affinity for the VVGAVGVGK-HLA A1101 complex, and the mutated residues are underlined.
  • Figures 9a and 9b respectively show the extracellular amino acid sequences of wild-type TCR ⁇ and ⁇ chains that can specifically bind to the VVGAVGVGK-HLA A1101 complex.
  • Figures 10a and 10b respectively show the amino acid sequences of wild-type TCR ⁇ and ⁇ chains that can specifically bind to the VVGAVGVGK-HLA A1101 complex.
  • Figure 11 is the binding curve of the soluble reference TCR, that is, the wild-type TCR and the VVGAVGVGK-HLA A1101 complex.
  • Figure 12 shows the results of the activation function experiment of effector cells transfected with high-affinity TCR of the present invention.
  • the present invention obtains a high-affinity T cell receptor (TCR) that recognizes the VVGAVGVGK short peptide, which is presented in the form of a VVGAVGVGK-HLA A1101 complex.
  • TCR high-affinity T cell receptor
  • the high-affinity TCR is in the 3 CDR regions of its ⁇ chain variable domain:
  • CDR3 ⁇ Mutation in ASLKGNNDMR; and/or in the 3 CDR regions of the ⁇ chain variable domain:
  • CDR3 ⁇ A mutation in ASSSSRWEQQF; after the mutation, the affinity and/or binding half-life of the TCR of the present invention for the VVGAVGVGK-HLA A1101 complex is at least twice that of the wild-type TCR.
  • TCR T cell receptor
  • the International Immunogenetics Information System can be used to describe TCR.
  • the natural ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain. Broadly speaking, each chain includes a variable region, a connecting region, and a constant region.
  • the beta chain usually also contains a short variable region between the variable region and the connecting region, but the variable region is often regarded as a part of the connecting region.
  • the unique IMGT TRAJ and TRBJ are used to determine the TCR connection region, and the IMGT TRAC and TRBC are used to determine the TCR constant region.
  • Each variable region contains 3 CDRs (complementarity determining regions), CDR1, CDR2, and CDR3, chimeric in the framework sequence.
  • the different numbers of TRAV and TRBV refer to different types of V ⁇ and V ⁇ , respectively.
  • the alpha chain constant domain has the following symbols: TRAC*01, where "TR” represents the T cell receptor gene; "A” represents the alpha chain gene; C represents the constant region; "*01” represents alleles Gene 1.
  • TRBC1*01 or TRBC2*01 where “TR” represents the T cell receptor gene; “B” represents the ⁇ -chain gene; C represents the constant region; “*01” represents the allele 1.
  • the constant region of the ⁇ chain is uniquely determined.
  • TCR ⁇ chain variable domain refers to the connected TRAV and TRAJ regions
  • TCR ⁇ chain variable domain refers to the connected TRBV and TRBD/TRBJ regions.
  • the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ ; the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ , respectively.
  • the framework sequence of the TCR variable domain of the present invention can be of murine or human origin, and is preferably of human origin.
  • the constant domain of TCR contains an intracellular part, a transmembrane region and an extracellular part.
  • the TCR of the present invention preferably does not include a transmembrane region.
  • the amino acid sequence of the TCR of the present invention refers to the extracellular amino acid sequence of the TCR.
  • the TCR sequence used in the present invention is of human origin.
  • the alpha chain amino acid sequence and the beta chain amino acid sequence of the "wild-type TCR" in the present invention are SEQ ID NO: 24 and SEQ ID NO: 25, respectively, as shown in Figures 10a and 10b.
  • the alpha chain amino acid sequence and the beta chain amino acid sequence of the "reference TCR" in the present invention are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b.
  • the extracellular amino acid sequence of the alpha chain and the extracellular amino acid sequence of the beta chain of the "wild-type TCR" in the present invention are SEQ ID NO: 22 and SEQ ID NO: 23, respectively, as shown in Figure 9a and Figure 9b.
  • amino acid sequences of the alpha and beta chain variable domains of the wild-type TCR capable of binding to the VVGAVGVGK-HLA A1101 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as shown in Fig. 1a and Fig. 1b.
  • polypeptide of the present invention TCR of the present invention
  • T cell receptor of the present invention are used interchangeably.
  • the position numbers of the amino acid sequence of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered in sequence from the N-terminal to the C-terminal.
  • TRBC1*01 or TRBC2*01 press from N
  • the 60th amino acid in the sequence from end to C end is P (proline)
  • Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention or it can be expressed as TRBC1* 01 or TRBC2*01 exon 1 of the 60th amino acid
  • the 61st amino acid is Q (glutamine) in sequence from N-terminal to C-terminal, then this
  • it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1, or it can be expressed as the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1, and so on.
  • the position numbers of the amino acid sequences of TRAV and TRBV in the variable regions are numbered according to the position numbers listed in IMGT.
  • the position number listed in IMGT is 46, it is described as the 46th amino acid of TRAV in the present invention, and the rest can be deduced by analogy.
  • the special instructions shall be followed.
  • tumor is meant to include all types of cancer cell growth or carcinogenic processes, metastatic tissues or malignant transformed cells, tissues or organs, regardless of the pathological type or the stage of infection.
  • tumors include, without limitation, solid tumors, soft tissue tumors, and metastatic lesions.
  • solid tumors include: malignant tumors of different organ systems, such as sarcoma, lung squamous cell carcinoma, and cancer.
  • sarcoma for example: infected prostate, lung, breast, lymph, gastrointestinal (for example: colon), and genitourinary tract (for example: kidney, epithelial cells), pharynx.
  • Lung squamous cell carcinoma includes malignant tumors, for example, most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, and esophageal cancer.
  • malignant tumors for example, most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, and esophageal cancer.
  • the above-mentioned metastatic lesions of cancer can also be treated and prevented by the method and composition of the present invention.
  • the ⁇ chain variable domain and ⁇ chain variable domain of TCR each contain 3 CDRs, which are similar to the complementarity determining regions of antibodies.
  • CDR3 interacts with short antigen peptides
  • CDR1 and CDR2 interact with HLA. Therefore, the CDR of the TCR molecule determines its interaction with the antigen short peptide-HLA complex.
  • the alpha chain variable domain amino acid sequence and the ⁇ chain variable domain amino acid sequence of the wild-type TCR that can bind the antigen short peptide VVGAVGVGK and HLA A1101 complex are SEQ ID NO:1 and SEQ, respectively ID NO: 2, this sequence is the first discovery by the inventor. It has the following CDR regions:
  • the present invention obtains a high-affinity TCR whose affinity with the VVGAVGVGK-HLA A1101 complex is at least twice the affinity of the wild-type TCR and the VVGAVGVGK-HLA A1101 complex by screening the above-mentioned CDR regions for mutations.
  • the present invention provides a T cell receptor (TCR), which has the activity of binding to the VVGAVGVGK-HLA A1101 complex.
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO:1, namely CDR1, CDR2, and CDR3 are located at positions 27-32, 50-55, and 90-99 of SEQ ID NO:1, respectively .
  • the amino acid residue numbering adopts the numbering shown in SEQ ID NO:1, 90A is the first A of CDR3 ⁇ , 91S is the second S of CDR3 ⁇ , and 92L is the third L and 93K of CDR3 ⁇ . It is the 4th K of CDR3 ⁇ .
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO: 2, namely CDR1, CDR2 and CDR3 are located at positions 27-31, 49-54 and 93 of SEQ ID NO: 2, respectively. -103 people. Accordingly, the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 2, 101Q is the 9th Q of CDR3 ⁇ , 102Q is the 10th Q of CDR3 ⁇ , and 103F is the 11th F of CDR3 ⁇ .
  • the TCR ⁇ chain variable domain after mutation includes one or more amino acid residues selected from the following group: 90G, 91V or 91I, 92M, 93L or 93M or 93Q, wherein the amino acid residue numbering adopts SEQ ID NO:1; and/or the TCR ⁇ chain variable domain after mutation includes one or more amino acid residues selected from the following group: 101P, 102M, 103V, wherein the amino acid residue numbering adopts SEQ ID NO: The number shown in 2.
  • the specific form of the mutation in the variable domain of the ⁇ chain includes one or several groups of A90G, S91V/I, L92M, and K93L/M/Q; the specific form of the mutation in the variable domain of the ⁇ chain Including one or several groups among Q101P, Q102M, and F103V.
  • the TCR ⁇ chain variable domain comprises three CDR regions, wherein the amino acid sequence of CDR1 ⁇ is DRVSQS; and the amino acid sequence of CDR2 ⁇ is IYSNGD.
  • the TCR ⁇ chain variable domain comprises three CDR regions, CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR3 ⁇ is [3 ⁇ X1][3 ⁇ X2][3 ⁇ X3][3 ⁇ X4]GNNDMR, and [3 ⁇ X1 ] Is A or G; and/or [3 ⁇ X2] is S or V or I; and/or [3 ⁇ X3] is L or M; and/or [3 ⁇ X4] is K or Q or L or M.
  • the TCR ⁇ chain variable domain comprises three CDR regions, wherein the amino acid sequence of CDR3 ⁇ is selected from the group consisting of ASLKGNNDMR, AVMQGNNDMR, GVLKGNNDMR, GVLLGNNDMR, GVLQGNNDMR, AILKGNNDMR, AVLKGNNDMR, AVLQGNNDMR, and AVLQGNNDMR.
  • the TCR ⁇ chain variable domain comprises three CDR regions, wherein the amino acid sequence of CDR1 ⁇ is SGHVS; and the amino acid sequence of CDR2 ⁇ is FQNEAQ.
  • the TCR ⁇ chain variable domain comprises three CDR regions, wherein the amino acid sequence of CDR3 ⁇ is: ASSSSRWEQQF or ASSSSRWEPMV.
  • the TCR of the present invention is an ⁇ heterodimeric TCR
  • the ⁇ chain variable domain of the TCR contains at least 90% of the amino acid sequence shown in SEQ ID NO:1; preferably, at least 92%; more preferably Ground, at least 94% (eg, can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) sequence homology
  • the ⁇ -chain variable domain of the TCR contains at least 90%, preferably at least 92%, and more preferably at least 94% (such as , It can be an amino acid sequence with at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology).
  • the TCR of the present invention is a single-chain TCR
  • the ⁇ -chain variable domain of the TCR contains at least 85%, preferably at least 90%, and more preferably at least the amino acid sequence shown in SEQ ID NO: 3; 92%; most preferably, at least 94% (eg, it can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% % Sequence homology); and/or the ⁇ -chain variable domain of the TCR contains at least 85% of the amino acid sequence shown in SEQ ID NO: 4, preferably at least 90%.
  • % more preferably, at least 92%; most preferably, at least 94%; (e.g., it can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% The sequence homology) of the sequence homology of the amino acid sequence.
  • the TCR comprises (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) and (ii) Both contain the variable domain and at least a part of the constant domain of the TCR chain.
  • the Thr48 of the wild-type TCR ⁇ chain constant region TRAC*01 exon 1 was mutated to cysteine, and the ⁇ chain constant region TRBC1*01 or TRBC2*01 exon 1
  • the Ser57 of Ser57 is mutated to cysteine to obtain the reference TCR. Its amino acid sequence is SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b, the cysteine residue after mutation Expressed in bold letters.
  • the above cysteine substitution can form artificial interchain disulfide bonds between the constant regions of the ⁇ and ⁇ chains of the reference TCR to form a more stable soluble TCR, which makes it easier to evaluate the complex of TCR and VVGAVGVGK-HLA A1101
  • the binding affinity and/or binding half-life between substances It should be understood that the CDR region of the TCR variable region determines its affinity with the pMHC complex. Therefore, the above-mentioned cysteine substitution in the TCR constant region does not affect the binding affinity and/or binding half-life of the TCR.
  • the measured binding affinity between the reference TCR and the VVGAVGVGK-HLA A1101 complex is considered to be the binding affinity between the wild-type TCR and the VVGAVGVGK-HLA A1101 complex.
  • the binding affinity between the TCR of the present invention and the VVGAVGVGK-HLA A1101 complex is at least 2 times that between the reference TCR and the VVGAVGVGK-HLA A1101 complex, it is equivalent to the TCR of the present invention and VVGAVGVGK.
  • the binding affinity between the -HLA A1101 complex is at least twice that between the wild-type TCR and the VVGAVGVGK-HLA A1101 complex.
  • the binding affinity (inversely proportional to the dissociation equilibrium constant K D ) and the binding half-life (expressed as T 1/2 ) can be determined by any suitable method. Such as the use of surface plasmon resonance technology for detection. It should be understood that doubling the affinity of TCR will cause K D to be halved. T 1/2 is calculated as In2 divided by the dissociation rate (K off ). Therefore, doubling T 1/2 will cause K off to be halved.
  • the same test protocol is used to detect the binding affinity or binding half-life of a given TCR several times, for example, 3 times or more, and the results are averaged.
  • the surface plasmon resonance (BIAcore) method in the examples herein is used to detect the affinity of the soluble TCR, and the conditions are: a temperature of 25° C. and a pH of 7.1-7.5.
  • This method detects that the dissociation equilibrium constant K D of the reference TCR to the VVGAVGVGK-HLA A1101 complex is 4.3E-06M, which is 4.3 ⁇ M.
  • the dissociation equilibrium of the wild-type TCR to the VVGAVGVGK-HLA A1101 complex is considered The constant K D is also 4.3 ⁇ M.
  • the affinity of the TCR and the VVGAVGVGK-HLA A1101 complex is at least twice that of the wild-type TCR.
  • the dissociation equilibrium constant KD of the TCR to the VVGAVGVGK-HLA A1101 complex is ⁇ 2.15 ⁇ M;
  • Any suitable method can be used for mutation, including but not limited to those based on polymerase chain reaction (PCR), cloning based on restriction enzymes, or ligation-independent cloning (LIC) methods.
  • PCR polymerase chain reaction
  • LIC ligation-independent cloning
  • the method for producing the TCR of the present invention can be, but is not limited to, screening a TCR with high affinity to the VVGAVGVGK-HLA A1101 complex from a diverse library of phage particles displaying such TCR, as shown in the literature (Li, et al( 2005) Nature Biotech 23(3):349-354).
  • genes expressing wild-type TCR alpha and beta chain variable domain amino acids or genes expressing slightly modified wild-type TCR alpha and beta chain variable domain amino acids can be used to prepare template TCRs.
  • the DNA encoding the variable domain of the template TCR then introduces the changes required to produce the high-affinity TCR of the present invention.
  • amino acid sequences of the ⁇ -chain variable domain and ⁇ -chain variable domain forming the heterodimeric TCR molecule are preferably from Table 1 below:
  • the TCR of the present invention is a part having at least one TCR ⁇ and/or TCR ⁇ chain variable domain. They usually contain both the TCR ⁇ chain variable domain and the TCR ⁇ chain variable domain. They can be ⁇ heterodimers or single-stranded forms or any other forms that can exist stably. In adoptive immunotherapy, the full-length chain of ⁇ heterodimeric TCR (including cytoplasmic and transmembrane domains) can be transfected.
  • the TCR of the present invention can be used as a targeting agent for delivering therapeutic agents to antigen-presenting cells or combined with other molecules to prepare bifunctional polypeptides to target effector cells. In this case, the TCR is preferably in a soluble form.
  • the prior art discloses that the introduction of artificial interchain disulfide bonds between the ⁇ and ⁇ chain constant domains of TCR can obtain soluble and stable TCR molecules, as described in patent document PCT/CN2015/093806 Narrated. Therefore, the TCR of the present invention may be a TCR in which an artificial interchain disulfide bond is introduced between the residues of the constant domain of its ⁇ and ⁇ chains. Cysteine residues form artificial interchain disulfide bonds between the alpha and beta chain constant domains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate positions in the natural TCR to form artificial interchain disulfide bonds.
  • Thr48 in TRAC*01 exon 1 and replacing Ser57 in TRBC1*01 or TRBC2*01 exon 1 to form a disulfide bond can also be: Thr45 of TRAC*01 exon 1 and TRBC1*01 or Ser77 of TRBC2*01 exon 1; TRAC*01 exon Tyr10 of 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 Ser15 and Glu15 of TRBC1*01 or TRBC2*01 exon 1; Arg53 of TRAC*01 exon 1 and Ser54 of TRBC1*01 or TRBC2*01 exon 1; Pro89 and Pro89 of TRAC*01 exon 1 Ala19 of TRBC1*01 or TRBC2*01 exon 1; or Tyr10 of TRAC*01 ex
  • cysteine residues replace any set of positions in the constant domains of the ⁇ and ⁇ chains.
  • One or more C-terminals of the TCR constant domain of the present invention can be truncated up to 15, or up to 10, or up to 8 or less amino acids so that it does not include cysteine residues to achieve the deletion of natural
  • the purpose of interchain disulfide bonds can also be achieved by mutating the cysteine residues that form natural interchain disulfide bonds to another amino acid.
  • the TCR of the present invention may contain artificial interchain disulfide bonds introduced between the residues of the constant domains of its ⁇ and ⁇ chains. It should be noted that, with or without the introduced artificial disulfide bonds between the constant domains, the TCR of the present invention can contain the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence.
  • the TRAC constant domain sequence of TCR and the TRBC1 or TRBC2 constant domain sequence can be linked by natural interchain disulfide bonds present in the TCR.
  • patent document PCT/CN2016/077680 also discloses that the introduction of artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region of the TCR can significantly improve the stability of the TCR. Therefore, the high-affinity TCR of the present invention may also contain artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region.
  • cysteine residue that forms an artificial interchain disulfide bond between the ⁇ chain variable region and the ⁇ chain constant region of the TCR is substituted: the 46th amino acid of TRAV and TRBC1*01 or TRBC2* The 60th amino acid of 01 exon 1; the 47th amino acid of TRAV and the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1; the 46th amino acid of TRAV and the TRBC1*01 or TRBC2*01 exon The 61st amino acid of sub 1; or the 47th amino acid of TRAV and the 60th amino acid of TRBC1*01 or TRBC2*01 exon 1.
  • such a TCR may comprise (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) and (ii) ) Contains the variable domain and at least a part of the constant domain of the TCR chain, and the ⁇ chain and the ⁇ chain form a heterodimer. More preferably, such a TCR may include an ⁇ chain variable domain and a ⁇ chain variable domain and all or part of the ⁇ chain constant domain except the transmembrane domain, but it does not include the ⁇ chain constant domain. The chain variable domain and the ⁇ chain form a heterodimer.
  • the TCR of the present invention also includes TCRs with mutations in the hydrophobic core region. These mutations in the hydrophobic core region are preferably mutations that can improve the stability of the TCR of the present invention, as described in Publication No. It is described in the patent document of WO2014/206304.
  • Such a TCR can be mutated at the following variable domain hydrophobic core positions: ( ⁇ and/or ⁇ chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/ Or alpha chain J gene (TRAJ) short peptide amino acid position from the bottom 3, 5, 7 and/or ⁇ chain J gene (TRBJ) short peptide amino acid position at the bottom 2, 4, 6 position, wherein the position number of the amino acid sequence According to the position number listed in the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the TCR whose hydrophobic core region is mutated in the present invention may be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the ⁇ and ⁇ chains of the TCR.
  • the CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex. Mutations in the hydrophobic core can make the TCR more stable, but it will not affect its affinity with the short peptide-HLA complex.
  • the flexible peptide chain in the present invention can be any peptide chain suitable for connecting the variable domains of the TCR ⁇ and ⁇ chains.
  • the template chain constructed in Example 1 of the present invention for screening high-affinity TCRs is the above-mentioned high-stability single-chain TCR containing a hydrophobic core mutation. Using TCR with higher stability can more conveniently evaluate the affinity between TCR and VVGAVGVGK-HLA A1101 complex.
  • the CDR regions of the ⁇ -chain variable domain and ⁇ -chain variable domain of the single-chain template TCR are exactly the same as the CDR regions of the wild-type TCR. That is, the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : DRVSQS, CDR2 ⁇ : IYSNGD, CDR3 ⁇ : ASLKGNNDMR, and the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : SGHVS, CDR2 ⁇ : FQNEAQ, and CDR3 ⁇ : ASSSSRWEQQF.
  • the amino acid sequence (SEQ ID NO: 9) and nucleotide sequence (SEQ ID NO: 10) of the single-stranded template TCR are shown in Figure 5a and Figure 5b, respectively. Based on this, a single-chain TCR composed of ⁇ -chain variable domain and ⁇ -chain variable domain with high affinity to the VVGAVGVGK-HLA A1101 complex was screened out.
  • the three CDRs of the single-chain template TCR ⁇ chain variable domain SEQ ID NO: 3, namely CDR1, CDR2, and CDR3 are located at positions 27-32, 50-55, and 90-99 of SEQ ID NO:1, respectively Bit. Accordingly, the amino acid residue numbering adopts the numbering shown in SEQ ID NO:1, 90A is the first A of CDR3 ⁇ , 91S is the second S of CDR3 ⁇ , and 92L is the third L and 93K of CDR3 ⁇ . It is the 4th K of CDR3 ⁇ .
  • the three CDRs of the single-stranded template TCR ⁇ chain variable domain SEQ ID NO: 4, namely CDR1, CDR2, and CDR3 are located at positions 27-31, 49-54 and 93-103 of SEQ ID NO: 2, respectively Bit. Accordingly, the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 2, 101Q is the 9th Q of CDR3 ⁇ , 102Q is the 10th Q of CDR3 ⁇ , and 103F is the 11th F of CDR3 ⁇ .
  • the ⁇ heterodimer with high affinity to the VVGAVGVGK-HLA A1101 complex of the present invention is obtained by transferring the CDR regions of the ⁇ and ⁇ chain variable domains of the screened high-affinity single-chain TCR to The wild-type TCR ⁇ chain variable domain (SEQ ID NO: 1) and ⁇ chain variable domain (SEQ ID NO: 2) are obtained from the corresponding positions.
  • the TCR of the present invention can also be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the present invention comprises a polymer formed by combining two, three, four or more TCRs of the present invention.
  • the tetramerization domain of p53 can be used to generate a tetramer, or more A complex formed by combining the TCR of the present invention with another molecule.
  • the TCR complex of the present invention can be used to track or target cells presenting a specific antigen in vitro or in vivo, and can also be used to produce intermediates of other multivalent TCR complexes with such applications.
  • the TCR of the present invention can be used alone, or can be combined with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugates include detectable markers (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the VVGAVGVGK-HLA A1101 complex), therapeutic agents, PK (protein kinase) modified parts or any of the above substances The combination of binding or coupling.
  • Detectable markers used for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or capable of producing detectable products Of enzymes.
  • Therapeutic agents that can be combined or coupled with the TCR of the present invention include but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al., 1989) , Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines such as IL-2, etc.
  • Gold Nanoparticles/Nano Stick (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 7. Virus particles (Peng et al., 2004, Gene Treatment (Genetherapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nano magnetic particles; 10. Prodrug activating enzymes (for example, DT-cardiac Diazyme (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. Chemotherapeutics (for example, cisplatin) or any form of nanoparticles, etc.
  • DTD DT-cardiac Diazyme
  • BPHL biphenyl hydrolase-like protein
  • the antibodies or fragments thereof that bind to the TCR of the present invention include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies.
  • the combination of the above-mentioned antibodies or fragments with TCR can affect effector cells. Orientation to better target target cells.
  • a preferred embodiment is that the TCR of the present invention is combined with an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody.
  • the fusion molecule of TCR and anti-CD3 single chain antibody of the present invention includes the amino acid sequence of the variable domain of the TCR ⁇ chain: one of SEQ ID NO: 13-20 and/or the amino acid sequence of the variable domain of the TCR ⁇ chain: SEQ ID NO: 2 , One of 21.
  • the invention also relates to a nucleic acid molecule encoding the TCR of the invention.
  • the nucleic acid molecule of the present invention may be in the form of DNA or RNA.
  • DNA can be a coding strand or a non-coding strand.
  • the nucleic acid sequence encoding the TCR of the present invention may be the same as the nucleic acid sequence shown in the drawings of the present invention or a degenerate variant.
  • degenerate variant refers to a protein sequence that encodes SEQ ID NO: 9, but is similar to the sequence of SEQ ID NO: 10 Different nucleic acid sequences.
  • the full-length sequence of the nucleic acid molecule of the present invention or its fragments can usually be obtained by but not limited to PCR amplification method, recombination method or artificial synthesis method.
  • the DNA sequence encoding the TCR (or a fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • the present invention also relates to a vector containing the nucleic acid molecule of the present invention, and a host cell produced by genetic engineering using the vector or coding sequence of the present invention.
  • the present invention also includes isolated cells expressing the TCR of the present invention, especially T cells.
  • T cells There are many methods suitable for T cell transfection with DNA or RNA encoding the high-affinity TCR of the present invention (eg, Robbins et al., (2008) J. Immunol. 180: 6116-6131).
  • T cells expressing the high-affinity TCR of the present invention can be used for adoptive immunotherapy.
  • Those skilled in the art can know many suitable methods for adoptive therapy (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).
  • the present invention also provides a pharmaceutical composition containing a pharmaceutically acceptable carrier and the TCR of the present invention, or the TCR complex of the present invention, or a cell presenting the TCR of the present invention.
  • the present invention also provides a method for treating diseases, comprising administering an appropriate amount of the TCR of the present invention, or the TCR complex of the present invention, or cells presenting the TCR of the present invention, or the pharmaceutical composition of the present invention to a subject in need of treatment.
  • A90G represents the replacement of A at position 90 by G
  • S91V/I represents the replacement of S at position 91 by V or I. The rest can be deduced by analogy.
  • the TCR of the present invention also includes at most 5 of the TCR of the present invention, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid (especially the amino acid located outside the CDR region), which are similar in nature. Or similar amino acids are replaced, and still maintain its functional TCR.
  • the present invention also includes a TCR slightly modified from the TCR of the present invention.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of the TCR of the present invention such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the TCR of the present invention or in further processing steps. This modification can be accomplished by exposing the TCR to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase).
  • Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). It also includes TCR that has been modified to improve its resistance to proteolysis or optimize its solubility.
  • the TCR, TCR complex of the present invention or T cells transfected with the TCR of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCR, multivalent TCR complex or cell of the present invention is usually provided as part of a sterile pharmaceutical composition, which usually includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be in any suitable form (depending on the desired method of administration to the patient). It can be provided in a unit dosage form, usually in a sealed container, and can be provided as part of a kit. Such kits (but not required) include instructions for use. It may include a plurality of such unit dosage forms.
  • the TCR of the present invention can be used alone, or can be combined or coupled with other therapeutic agents (for example, formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent.
  • pharmaceutical carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the composition, and do not have excessive toxicity after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Such carriers include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain liquids such as water, saline, glycerol and ethanol.
  • these carriers may also contain auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances.
  • the therapeutic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • an injectable such as a liquid solution or suspension
  • it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • composition of the present invention can be administered by conventional routes, including (but not limited to): intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal External includes subcutaneous, intramuscular or intravenous.
  • routes including (but not limited to): intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal External includes subcutaneous, intramuscular or intravenous.
  • the objects to be prevented or treated can be animals; especially humans.
  • composition of the present invention When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the use situation. Preferably, injections, oral preparations and the like can be exemplified.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a customary manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a customary manner according to the dosage form.
  • the pharmaceutical composition of the present invention can also be administered in the form of a sustained-release dosage form.
  • the TCR of the present invention can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the TCR or TCR complex of the present invention as the active ingredient or the cells presenting the TCR of the present invention can be based on the weight, age, sex, and degree of symptoms of each patient to be treated. It is determined reasonably, and the doctor finally decides the reasonable dosage.
  • the affinity and/or binding half-life of the TCR of the present invention for the VVGAVGVGK-HLA A1101 complex is at least twice that of the wild-type TCR.
  • the effector cells that transduce the high-affinity TCR of the present invention have a strong activation function on target cells.
  • E. coli DH5 ⁇ was purchased from Tiangen
  • E. coli BL21 (DE3) was purchased from Tiangen
  • E. coli Tuner (DE3) was purchased.
  • plasmid pET28a was purchased from Novagen.
  • Example 1 Stability of hydrophobic core mutations. Generation of single-stranded TCR template strands
  • the present invention uses the method of site-directed mutagenesis, according to the patent document WO2014/206304, to construct a stable single-stranded TCR molecule composed of a flexible short peptide (linker) connecting TCR ⁇ and ⁇ chain variable domains, its amino acids and DNA
  • the sequences are SEQ ID NO: 9 and SEQ ID NO: 10, respectively, as shown in Figure 5a and Figure 5b.
  • the amino acid sequences of the ⁇ variable domain (SEQ ID NO: 3) and ⁇ variable domain (SEQ ID NO: 4) of the template chain are shown in Figure 2a and Figure 2b; the corresponding DNA sequences are respectively SEQ ID NO: 5 and 6, as shown in Figure 3a and Figure 3b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are SEQ ID NO: 7 and 8, respectively, as shown in Figure 4a and Figure 4b.
  • the target gene carrying the template chain was digested with NcoI and NotI, and then connected to the pET28a vector that was digested with NcoI and NotI.
  • the ligation product was transformed into E.coli DH5 ⁇ , spread on an LB plate containing kanamycin, incubated overnight at 37°C, and selected positive clones for PCR screening, and sequenced the positive recombinants to confirm the correct sequence and extract the recombinant plasmid for transformation To E.coli BL21(DE3), for expression.
  • Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1
  • a syringe to drop the single-stranded TCR treated above into 125mL of refolding buffer (100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine), Stir at 4°C for 10 minutes, then put the refolding solution into a cellulose membrane dialysis bag with a cutoff of 4kDa, place the dialysis bag in 1L of pre-cooled water, and slowly stir overnight at 4°C.
  • refolding buffer 100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine
  • the collected elution fractions were analyzed by SDS-PAGE, and the fractions containing single-stranded TCR were concentrated and further purified with a gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.
  • the eluted fractions used for BIAcore analysis were further tested for purity by gel filtration.
  • the conditions are: Column Agilent Bio SEC-3 (300A, ), the mobile phase is 150mM phosphate buffer, the flow rate is 0.5mL/min, the column temperature is 25°C, and the UV detection wavelength is 214nm.
  • the BIAcore T200 real-time analysis system was used to detect the binding activity of the TCR molecule and the VVGAVGVGK-HLA A1101 complex.
  • the conditions are: the temperature is 25°C and the PH value is 7.1-7.5.
  • the binding time of each injection is 120s, and let it dissociate for 600s after the last injection.
  • the chip was regenerated with 10mM Gly-HCl pH 1.75. Use BIAcore Evaluation software to calculate kinetic parameters.
  • the synthetic short peptide VVGAVGVGK (Beijing Saibaisheng Gene Technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml.
  • the inclusion bodies of the light chain and the heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA were added before renaturation to further denature.
  • VVGAVGVGK short peptide at 25mg/L (final concentration) into the refolding buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione) , 0.2mM PMSF, cooled to 4°C), then add 20mg/L of light chain and 90mg/L of heavy chain (final concentration, heavy chain is added in three times, 8h/time), renaturation is carried out at 4°C for at least 3 After the day is over, SDS-PAGE will test whether the refolding is successful.
  • the refolding buffer 0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione
  • PMSF cooled to 4°C
  • renaturation is carried out at 4°C for at least 3 After the day is over, SDS-PAGE will test whether
  • the protein-containing fractions were combined, concentrated with a Millipore ultrafiltration tube, and the protein concentration was determined by the BCA method (Thermo), and the protease inhibitor cocktail (Roche) was added to store the biotinylated pMHC molecules in aliquots at -80°C.
  • Phage display technology is a means to generate a library of TCR high-affinity variants to screen high-affinity variants.
  • the TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3):349-354) was applied to the single-stranded TCR template in Example 1.
  • a high-affinity TCR library was established and panned. After several rounds of panning, the phage library has specific binding to the corresponding antigen, and a single clone is selected from it, and sequence analysis is performed.
  • the screened high-affinity single-chain TCR mutations in the CDR region were introduced into the corresponding sites of the variable domain of the ⁇ heterodimeric TCR, and the affinity with the VVGAVGVGK-HLA A1101 complex was detected by BIAcore.
  • the introduction of the above-mentioned high-affinity mutation points in the CDR region adopts a site-directed mutation method well known to those skilled in the art.
  • the amino acid sequences of the alpha chain and beta chain variable domains of the wild-type TCR are shown in Figure 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2), respectively.
  • the ⁇ heterodimeric TCR can be constant in the ⁇ and ⁇ chains.
  • a cysteine residue is introduced into the regions to form the TCR of the artificial inter-chain disulfide bond.
  • the amino acid sequences of the TCR ⁇ and ⁇ chains after the introduction of cysteine residues are shown in Figure 6a (SEQ ID NO : 11) and shown in 6b (SEQ ID NO: 12), the introduced cysteine residues are indicated in bold letters.
  • the extracellular sequence genes of the TCR ⁇ and ⁇ chains to be expressed were synthesized and inserted into the expression vector by the standard method described in the "Molecular Cloning a Laboratory Manual” (third edition, Sambrook and Russell) For pET28a+ (Novagene), the upstream and downstream cloning sites are NcoI and NotI, respectively. Mutations in the CDR region are introduced by overlapping PCR (overlap PCR), which is well known to those skilled in the art. The inserted fragment was confirmed by sequencing.
  • TCR ⁇ and ⁇ chains were respectively transformed into expressing bacteria BL21(DE3) by chemical transformation method.
  • the ⁇ and ⁇ chains of TCR were expressed
  • the inclusion bodies formed later were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution.
  • the inclusion bodies were finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA) ), 20mM Tris (pH 8.1).
  • the dissolved TCR ⁇ and ⁇ chains are quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C) at a mass ratio of 1:1, and the final concentration is 60mg/mL.
  • 5M urea 20mM Tris (pH 8.1)
  • 20mM Tris 20mM Tris (pH 8.1)
  • cystamine 3.7mM cystamine
  • 6.6mM ⁇ -mercapoethylamine 4°C
  • the solution is filtered through a 0.45 ⁇ M filter membrane and purified by an anion exchange column (HiTrap Q HP, 5ml, GE Healthcare).
  • the eluted peak contains the successfully renatured ⁇ and ⁇ dimer TCRs, which are confirmed by SDS-PAGE gel.
  • the TCR is then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • Example 3 The method described in Example 3 was used to detect the affinity of the ⁇ heterodimeric TCR introduced into the high-affinity CDR region and the VVGAVGVGK-HLA A1101 complex.
  • the present invention obtains the high-affinity TCR alpha chain and beta chain variable domain amino acid sequences, as shown in Figure 7a to Figure 7h and Figure 8 respectively. Since the CDR region of the TCR molecule determines its affinity with the corresponding pMHC complex, those skilled in the art can expect that the ⁇ heterodimeric TCR introduced with a high-affinity mutation point will also have a high affinity for the VVGAVGVGK-HLA A1101 complex.
  • Example 4 Use the method described in Example 4 to construct an expression vector, use the method described in Example 5 to express, renature and purify the ⁇ heterodimeric TCR introduced with high affinity mutations, and then use BIAcore T200 to determine its relationship with VVGAVGVGK-
  • Table 2 The affinity of HLA A1101 complex is shown in Table 2 below.
  • the affinity of the high-affinity TCR obtained in the present invention is at least twice the affinity of the wild-type TCR for the VVGAVGVGK-HLA A1101 complex.
  • Example 7 Expression, renaturation and purification of the fusion of anti-CD3 antibody and high-affinity ⁇ heterodimeric TCR
  • the anti-CD3 single-chain antibody (scFv) is fused with ⁇ heterodimeric TCR to prepare a fusion molecule.
  • the anti-CD3 scFv is fused with the ⁇ chain of the TCR, and the TCR ⁇ chain may include any of the above-mentioned high-affinity ⁇ heterodimeric TCR ⁇ -chain variable domains, and the TCR ⁇ chain of the fusion molecule may include any of the above-mentioned high affinity
  • the target gene carrying the ⁇ chain of ⁇ heterodimeric TCR was digested with NcoI and NotI, and then connected to the pET28a vector that was digested with NcoI and NotI.
  • the ligation product was transformed into E.coli DH5 ⁇ , spread on an LB plate containing kanamycin, and incubated overnight at 37°C. Positive clones were selected for PCR screening, the positive recombinants were sequenced, and the recombinant plasmids were extracted after the sequence was confirmed. Transform into E.coli Tuner (DE3) for expression.
  • primers are designed to link the anti-CD3 scFv and the high-affinity heterodimeric TCR ⁇ chain gene, the linker in the middle is GGGGS, and the anti-CD3 scFv is connected to the high-affinity heterodimeric TCR ⁇ chain gene.
  • the gene fragment of the fusion protein of the affinity heterodimeric TCR ⁇ chain carries restriction endonuclease sites NcoI(CCATGG) and NotI(GCGGCCGC).
  • the PCR amplified product was digested with NcoI and NotI, and then ligated with the pET28a vector that was digested with NcoI and NotI.
  • the ligation product was transformed into E.coli DH5 ⁇ competent cells, spread on LB plates containing kanamycin, incubated overnight at 37°C, picked positive clones for PCR screening, sequenced the positive recombinants, and extracted after confirming the correct sequence
  • the recombinant plasmid is transformed into E. coli Tuner (DE3) competent cells for expression.
  • the expression plasmids were respectively transformed into E. coli Tuner (DE3) competent cells, spread on LB plates (kanamycin 50 ⁇ g/mL) and incubated overnight at 37°C. On the next day, pick the clones and inoculate them into 10mL LB liquid medium (kanamycin 50 ⁇ g/mL) for 2-3 hours, inoculate them into 1L LB medium at a volume ratio of 1:100, continue to cultivate until the OD600 is 0.5-0.8, add The final concentration is 1mM IPTG induces the expression of the target protein. After 4 hours of induction, the cells were harvested by centrifugation at 6000 rpm for 10 min. Wash the cells once with PBS buffer and separate the cells.
  • the dissolved TCR ⁇ chain and anti-CD3 (scFv)- ⁇ chain are quickly mixed with 5M urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH 8.1, 3.7 at a mass ratio of 2:5 mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C), the final concentration of ⁇ chain and anti-CD3 (scFv)- ⁇ chain are 0.1mg/mL and 0.25mg/mL, respectively.
  • the TCR fusion molecule is then further purified by size exclusion chromatography (S-100 16/60, GE healthcare), and again purified by anion exchange column (HiTrap Q HP 5ml, GE healthcare).
  • the purity of the purified TCR fusion molecule was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • Example 8 Activation function experiment of effector cells transfected with high-affinity TCR of the present invention
  • the CD3+ T cells isolated from the blood of healthy volunteers were randomly selected to be transfected with the TCR of the present invention as effector cells.
  • the TCR and its number are learned from Table 2.
  • TCR1 ⁇ chain variable domain SEQ ID NO: 13, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR3 ⁇ chain variable domain SEQ ID NO: 15, ⁇ chain variable domain SEQ ID NO: 2)
  • the control effector cells are labeled wild-type TCR (cells transfected with wild-type TCR) and A6 (cells transfected with other TCRs).
  • K562-A11-TMG1 (A11 and KRAS G12V overexpression), K562-A11 (A11 overexpression), K562-A11-TMG2 (A11 and KRAS G12D overexpression), SK-MEL-1, SW620-A11 (A11 overexpression)
  • the target cell lines K562-A11-TMG1 and SW620-A11 were used as positive tumor cell lines; K562-A11, K562-A11-TMG2, SK-MEL-1 and SW620 were negative tumor cell lines, which were used as controls.
  • ELISPOT plate was activated and coated with ethanol at 4°C overnight. On the first day of the experiment, remove the coating solution, wash and block, incubate for two hours at room temperature, remove the blocking solution, and add the test components to the ELISPOT plate: target cells are 2 ⁇ 10 4 cells/well, and effector cells are 10 3 Pcs/well (calculated according to the transfection positive rate), and set up two duplicate wells. Incubate overnight (37°C, 5% CO 2 ). On the second day of the experiment, the plate was washed and subjected to secondary detection and color development. The plate was dried, and then the spots formed on the membrane were counted with an immunospot plate reader (ELISPOT READER system; AID20 company).
  • ELISPOT READER system an immunospot plate reader
  • the effector cells transfected with the high-affinity TCR of the present invention produced a very good specific activation effect, and did not respond to the negative target cell line.
  • effector cells that transduce other TCRs basically have no activation effect.

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Abstract

La présente invention concerne un récepteur de lymphocytes T (TCR) ayant la caractéristique de se lier à un complexe VVGAVGVGK-HLA A1101. L'affinité de liaison du TCR au complexe VVGAVGVGK-HLA A1101 est au moins 2 fois celle d'un TCR de type sauvage au complexe VVGAVGVGK-HLA A1101. La présente invention concerne également une molécule de fusion d'un tel TCR avec un agent thérapeutique. Un tel TCR peut être utilisé seul ou en combinaison avec l'agent thérapeutique pour cibler des cellules tumorales présentant le complexe VVGAVGVGK-HLA A1101.
PCT/CN2020/125475 2019-11-01 2020-10-30 Tcr à haute affinité pour la reconnaissance de kras g12v Ceased WO2021083363A1 (fr)

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WO2023102418A1 (fr) * 2021-12-01 2023-06-08 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs de lymphocytes t à restriction hla-a3 contre ras avec mutation g12v
WO2023139257A1 (fr) * 2022-01-21 2023-07-27 T-Knife Gmbh Construction reconnaissant un antigène se liant à un peptide spécifique avec une affinité déterminable et récepteur de lymphocytes t ayant une spécificité antigénique pour kras, séquence d'acide nucléique correspondante, vecteur, cellule hôte, composition pharmaceutique et kit
WO2023220718A1 (fr) * 2022-05-13 2023-11-16 Fred Hutchinson Cancer Center Protéines de liaison spécifiques pour des néo-antigènes ras et leurs utilisations
WO2024093056A1 (fr) * 2022-11-04 2024-05-10 新景智源生物科技(苏州)有限公司 Tcr spécifique d'un antigène mutant kras_g12v et lymphocyte t cd4 redirigé co-exprimant celui-ci et cd8
WO2024149347A1 (fr) * 2023-01-13 2024-07-18 北京可瑞生物科技有限公司 Protéine de liaison à l'antigène et son utilisation
WO2024245375A1 (fr) * 2023-06-01 2024-12-05 皖南医学院第一附属医院（皖南医学院弋矶山医院） Fragment d'anticorps à chaîne unique ciblant kras g12v, car de récepteur antigénique chimérique et utilisations
WO2025176000A1 (fr) * 2024-02-20 2025-08-28 上海信谱生物医药科技有限公司 Récepteur de lymphocytes t pour identifier une mutation kras et séquence de codage associée

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CN116063511B (zh) * 2021-09-30 2023-10-03 北京可瑞生物科技有限公司 抗原结合蛋白及其应用
CN117264043B (zh) * 2022-06-14 2024-05-10 上海镔铁生物科技有限责任公司 靶向kras g12v突变多肽的t细胞受体及其用途

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CN108350059A (zh) * 2015-09-15 2018-07-31 美国卫生和人力服务部 识别hla-cw8限制性突变kras的t细胞受体
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023102418A1 (fr) * 2021-12-01 2023-06-08 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs de lymphocytes t à restriction hla-a3 contre ras avec mutation g12v
WO2023139257A1 (fr) * 2022-01-21 2023-07-27 T-Knife Gmbh Construction reconnaissant un antigène se liant à un peptide spécifique avec une affinité déterminable et récepteur de lymphocytes t ayant une spécificité antigénique pour kras, séquence d'acide nucléique correspondante, vecteur, cellule hôte, composition pharmaceutique et kit
WO2023220718A1 (fr) * 2022-05-13 2023-11-16 Fred Hutchinson Cancer Center Protéines de liaison spécifiques pour des néo-antigènes ras et leurs utilisations
WO2024093056A1 (fr) * 2022-11-04 2024-05-10 新景智源生物科技(苏州)有限公司 Tcr spécifique d'un antigène mutant kras_g12v et lymphocyte t cd4 redirigé co-exprimant celui-ci et cd8
WO2024149347A1 (fr) * 2023-01-13 2024-07-18 北京可瑞生物科技有限公司 Protéine de liaison à l'antigène et son utilisation
WO2024245375A1 (fr) * 2023-06-01 2024-12-05 皖南医学院第一附属医院（皖南医学院弋矶山医院） Fragment d'anticorps à chaîne unique ciblant kras g12v, car de récepteur antigénique chimérique et utilisations
WO2025176000A1 (fr) * 2024-02-20 2025-08-28 上海信谱生物医药科技有限公司 Récepteur de lymphocytes t pour identifier une mutation kras et séquence de codage associée

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